The normal structure and function of adult organs is maintained by resident stem cells and their progeny throughout life. The proliferation of adult stem cells and differentiation of their progeny must be tightly controlled. One of the best examples is the adult gastrointestinal epithelium which is constantly being renewed with the progeny of ISCs. ISCs replenish the intestinal epithelium every few days and represent a general model for tissue differentiation and stem cell properties. Deregulation of the regulatory mechanisms could result in the depletion of stem cells or in the excessive proliferation of stem cells/progenitor cells, eventually leading to diseases such as cancer. The long-term goal of this proposal is to elucidate the molecular mechanism(s) by which adult ISC activities and homeostasis are regulated. We will use Drosophila adult midgut as a system. Over the past 7 years, studies in this system have demonstrated that the JAK/Stat, Dpp (Drosophila BMP), EGFR, and Hippo signaling pathways are required for midgut homeostasis under normal physiological conditions or in response to environmental challenges. However, currently it is unknown how these signaling pathways execute their signaling activities through their downstream target genes to control gut homeostasis. In our recent studies, we have demonstrated that BMP pathway, Hh signaling pathway, and HSPGs can regulate stem cell proliferation, differentiation, and stem cell-niche interactions in different manners, and play essential roles in regulating stem cell fate and homeostasis. Moreover, we have conducted ChIP- Seq experiments combined with RNAi-mediated screens to identify JAK/STAT downstream target genes. On the basis of our recent published studies and our unpublished data, we have proposed a number of important hypotheses aimed at elucidation of the molecular mechanisms regulating ISC activities and homeostasis. In this proposal, we will use Drosophila adult midgut as a system to address three independent, but highly related issues in the maintenance of gut homeostasis. First, we will examine mechanism(s) of the JAK/STAT signaling pathway in regulating gut homeostasis. Second, we will define the mechanism(s) of Dpp signaling in regulating stem cell activity by mediating tracheal-gut interactions. Third, we will examine the mechanisms of Perlecan/ECM in the regulation of ISC-niche interactions. Taken together, integration of these three important regulatory mechanisms will enhance our understanding of the maintenance of homeostasis, signaling transduction, and stem cell regulation. The outcomes of this will shed light on human diseases associated with deregulation of ISC activities and gut homeostasis.